Our results in cultured human endometrial cells indicate that E2 binding levels can double or triple within two hours and decrease just as rapidly. We propose to continue our investigation into the biochemical mechanism responsible for such rapid changes in binding and to try to determne their physiological importance in the overall regulation of E2 hormone action. Using Na2Mo04, we have found methods to increase E2 binders in intact cells and to activate E2 binding sites in cellular homogenates. In addition to Mo04, we have discovered that physiologically important compounds such as ATP, GTP and CGMP can activate and cAMP can inactivate E2 binding sites. We plan to do further experiments in cellular homogenates to determine the mechanism involved in E2 binder regulation and the roles ot ATP, GTP, and cAMP. Perliminary studies will be done to look for evidence of phosphorylation and dephosphorylation as a possible cause of binder activation/inactivation. From studies of the Mo04 effect on activation, it appears that cytoplasmic and membrane associated factors are required. We will try to characterize these factors and determine if they are also necessary for ATP, GTP, cGMP, and cAMP effects. Under our assay conditions, estriol remains associated only with the higher affinity binder and will be used in parallel binding studies with E2 to determine whether ATP, GTP, and cGMP increase both binders as does Mo04 and whether both or only one binder is capable of translocation. We will try to relate observation on E2 binding activation and inactivation in cellular homogenates to binder fluctuations in intact cells. We will try to determine how levels of binder available for activation are regulated in intact cells. The various studies we have described will be done in HEC cells, in normal endometrium, and in endometrial tumors. We are particulatly interested in determining whether some tumors classified as receptor negative may not actually contain inactive binders.